Method for purifying calcium ion-binding protein

ABSTRACT

The present invention relates to a method for purifying a calcium ion-binding protein by cation exchange chromatography. The present invention provide a method for isolating and purifying a calcium ion-binding protein in a simple and efficient manner from a liquid sample containing a calcium ion-binding protein and contaminants without any pretreatment such as addition of a chelating agent. More specifically, the present invention relates to a method for purifying a calcium ion-binding protein which comprises contacting said protein with a cation exchange carrier in the presence of calcium ions to let the said protein be adsorbed to the carrier, and after washing, eluting said protein, and to a calcium ion-binding protein having substantially no contaminants obtained by the method of the present invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for purifying a calciumion-binding protein by a cation exchange process. More specifically, thepresent invention relates to a method for purifying a calciumion-binding protein which comprises contacting said protein with acation exchange carrier in the presence of calcium ions to render saidprotein be adsorbed to the cation exchange carrier, and after washing,eluting said protein from the cation exchange carrier, and to a calciumion-binding protein obtained by said method which contains substantiallyno contaminants.

BACKGROUND OF THE INVENTION

For isolation and purification of a protein of interest fromcontaminants, physico-chemical properties such as a molecular size, anelectric charge on the surface or solubility of said protein isutilized. A process for purification commonly used in the field ofprotein chemistry includes, for instance, salting out, ultrafiltration,isoelectric precipitation, electrophoresis, ion exchange chromatography,gel filtration chromatography, affinity chromatography, and the like. Incase that a protein of interest must be purified from the livingtissues, cells, or blood, where an enormous variety of differentproteins exist, these processes need often be combined in a manifoldmanner. However, it is possible to provide a method for purificationwith much more specificity by utilizing a property commonly shared by acertain kind of protein.

By way of example, a unique method for purification using anion exchangechromatography is known wherein a divalent cation-binding protein isadsorbed to an anion exchange resin and then eluted therefrom with adivalent cation to specifically purify said divalent cation-bindingprotein as disclosed in Japanese patent publication No. 200180/1990.According to this method, a chelating reagent such asethylenediaminetetraacetic acid (EDTA) is added to a solution containinga divalent cation-binding protein to first remove divalent ions. Then,the resulting solution is contacted with an anion exchange resin such asMonoQ to render the divalent cation-binding protein be adsorbed to theanion exchange resin. Finally, addition of sodium chloride and calciumchloride elutes the divalent cation-binding protein from the anionexchange resin. However, most of naturally occurring proteins arenegatively charged under physiological conditions and hence numerouscontaminants other than a protein of interest are preferentiallyadsorbed to an anion exchange resin, thus hampering efficientpurification of the desired protein. Therefore, this method forpurification through adsorption of a desired protein to an anionexchange resin is preferably used for a small amount of a proteinsolution or at an advanced stage of purification processes.

Japanese patent publication No. 258286/1995 discloses a method forpurifying a calcium ion-binding, vitamin K-dependent protein by an anionexchange process wherein calcium chloride is added to a solutioncontaining a vitamin K-dependent protein and the resulting solution ispassed through an anion exchange resin to isolate the desired proteinfrom contaminating proteins. This method, however, is disadvantageous inthat a large volume of fractions containing the desired protein must bepassed through and hence subsequent procedures will become troublesomeespecially when conducted in a large scale.

Annexin V, one of calcium ion-binding proteins, is a simple protein ofabout 34 kDa molecular weight bearing no sugar chain that has aphysiological activity such as anti-coagulating activity, cornealepithelium-extending activity, and phospholipase A₂ inhibitory activity.It is known that Annexin V distributes in a variety of tissues andsecretions within the living body including human placenta (Chem.Pharma. Bull., 38, 1957-1960, 1990). Annexin V is called a calciumion-binding protein since it has an ability to bind with a lipidmembrane via calcium ions.

Annexin V has been extracted from organs of human or animals (Japanesepatent publication No. 174023/1987). Nowadays, however, it can beproduced in E. coli and yeast by the use of the genetic recombinationtechnique (Japanese patent publications No. 20095/1989 and No.219875/1991).

Annexin V has conventionally been purified, after pretreatment of anAnnexin V-containing solution with precipitation, membrane filtrationand centrifugation, by a combination of ammonium sulfate fractionation,anion exchange chromatography, hydrophobic chromatography and affinitychromatography (Jurgen Romisch et al., Biochem. J. 272, 223-229, 1990;T. R. Hawthorne et al., Journal of Biotechnology 36, 129-143, 1994).

DISCLOSURE OF THE INVENTION

However, these processes are disadvantageous in that purification stepsare complicated and troublesome requiring a great deal of labor and timeand hence possibly meet an obstacle in view of reproducibility andyield, rendering them not be suitable for purification of Annexin V inan industrial scale. Moreover, as purification process in a large scale,these processes are disadvantageous in economical point of view as wellsince they used heparin Sepharose, which is rather expensive, forenhancing purification degree of Annexin V. Previously, the presentinventors have provided a method for preparing Annexin V by pretreatinga protein solution to remove contaminants to some extent and thenperforming anion exchange chromatography on the resulting solution(Japanese patent publication No. 219875/1991). This method mightpossibly enables purification of Annexin V in an industrial scale butwould not exceed the method of the present invention.

As described above, for use in an industrial scale, the conventionalprocesses are problematic in view of cost, efficiency and handling.

An object of the present invention is to provide a method for isolatingand purifying a calcium ion-binding protein in a simple and efficientmanner from a liquid sample containing a calcium ion-binding protein andcontaminants without any pretreatment such as addition of a chelatingagent.

Another object of the present invention is to provide Annexin V of highpurity obtained by the method of the present invention.

Under the circumstances, the present inventors investigated forattaining the above objects and have found that Annexin V, one ofcalcium ion-binding proteins, is adsorbed to SP-Sepharose cationexchange carrier in the presence of calcium chloride and at pH of aroundneutrality. The present inventors have also noted that the adsorption ofAnnexin V to SP-Sepharose cation exchange carrier occurred specificallyin the presence of calcium ions but could scarcely observe theadsorption in the presence of other divalent ions than calcium ions,e.g. magnesium ions. With this finding, the present inventors addedcalcium chloride to a large amount of homogenate of Annexin V-producingcells produced by the genetic recombination technique and contacted theresulting homogenate with SP-Sepharose cation exchange carrier which hasbeen equilibrated with ammonium chloride buffer containing calciumchloride. After washing, elution was performed by decreasing or removingcalcium chloride level or with ammonium chloride buffer containingsodium chloride in the presence of calcium ions to successfully purifyAnnexin V with high purity. Moreover, a trace amount of remainingproteases could successfully be removed by performing said cationexchange chromatography at pH 9.0.

Thus, the present invention encompasses a method for purification of acalcium ion-binding protein, either naturally occurring or produced bythe genetic recombination technique, by cation exchange chromatographyusing SP-Sepharose cation exchange carrier in the presence of calciumchloride.

The present invention also encompasses a calcium ion-binding protein,either naturally occurring or produced by the genetic recombinationtechnique, thus obtained by the method of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a schematic illustration of cloning of Annexin Vstructural gene and preparation of yeast cells transformed with saidgene.

FIG. 2 shows results of gel filtration chromatography for (a) samplesprior to cation exchange chromatography, (b) fractions passed throughcation exchange chromatography, and (c) fractions eluted from cationexchange chromatography after washing.

FIG. 3 shows results of gel filtration chromatography for fractionseluted from cation exchange chromatography after washing.

FIG. 4 shows an elution pattern of Annexin VI from SP-Sepharose.

FIG. 5 is a photograph showing results of SDS-PAGE of Annexin VI. Lane1: BioRad prestained marker proteins; phosphorylase B (116,000), BSA(80,000), ovalbumin (52,500), carbonic anhydrase (34,900), soybeantrypsin inhibitor (29,900), lysozyme (21,800); Lane 2: Annexin VIstandard; Lane 3: elution with DEAE-Toyopearl (sample); Lane 4:SP-Sepharose, fraction No. 5; Lane 5: fraction No. 7; Lane 6: fractionNo. 9; Lane 7: fraction No. 16; Lane 8: fraction No. 51; Lane 9:fraction No. 54; and Lane 10: fraction No. 57.

FIG. 6 shows an elution pattern of coagulation factor X fromSP-Sepharose.

FIG. 7 is a photograph showing results of SDS-PAGE of coagulation factorX. Lane 1: BioRad prestained marker proteins; phosphorylase B (116,000),BSA (80,000), ovalbumin (52,500), carbonic anhydrase (34,900), soybeantrypsin inhibitor (29,900), lysozyme (21,800); Lane 2: commerciallyavailable coagulation factor X; Lane 3: fraction No. 4; Lane 4: fractionNo. 7; Lane 5: fraction No. 11; and Lane 6: fraction No. 12.

BEST MODE FOR CARRYING OUT THE INVENTION

The method of the present invention comprises a step in which a liquidsample containing a calcium ion-binding protein is contacted with acation exchange carrier in the presence of calcium ion, followed by astep in which a concentration of calcium ion is decreased or removedand/or a concentration of counter ions (salts) is increased to elute andrecover said protein. The method of the present invention enablesproduction of a calcium ion-binding protein with high purity. Thecontacting process with the cation exchange carrier may be performedeither in a batch or by chromatography. When chromatography is used, acolumn size may appropriately be selected depending on a productionscale.

A cation exchange carrier used herein includes, but not limited to,SP-Sepharose, CM-Sepharose, CM-cellulose, SE-cellulose, S-Spherodex,SP-Spherosil, and the like, all of which are commercially available.Among these, SP-Sepharose is preferably used.

An amount of a protein solution to be contacted with the carrier mayvary depending on a concentration of the solution or an ability of thecarrier for adsorption. In case of SP-Sepharose, for instance, 0.1 to 30g/L carrier of the protein may be used. Preferably, 15 to 20 g/L carrierof the protein is used.

A flow rate while adsorption to the cation exchange carrier may be 1 to150 cm/h, preferably 15 to 100 cm/h, more preferably 50 to 80 cm/h. Onthe other hand, a flow rate while elution of the adsorbed protein fromthe cation exchange carrier may be 1 to 150 cm/h, preferably 30 to 100cm/h, more preferably 30 to 80 cm/h.

A buffer that may be used for adsorption to or elution from the cationexchange carrier includes any buffer conventionally used in ion exchangechromatography, including ammonium chloride buffer, citrate buffer,acetate buffer and Tris-HCl buffer. Among these, ammonium chloridebuffer is preferably used. A concentration of a buffer may be in a rangeof 5 to 100 mM, preferably 10 to 40 mM. A buffer may be used at pH 5 to10, preferably at pH 8 to 9.5, conditions where proteases are removed.More preferably, 20 mM (around pH 9.0) ammonium chloride buffer is used.

As a source of calcium ions, any substance that can afford calcium ionsmay be used, including calcium chloride, calcium carbonate, preferablycalcium chloride.

When a large quantity of calcium chloride is added to homogenate oftissues or cells or plasma, hydrophobic proteins or high molecularweight compounds are sometimes deposited as a result of salting-out.Thus, calcium ions may preferably be used in such an amount that notonly renders a calcium ion-binding protein be bound to and isolated fromthe cation exchange carrier but also forms no precipitation from aliquid sample containing a calcium ion-binding protein.

For adsorption of a calcium ion-binding protein to the cation exchangecarrier, calcium ions at a concentration of 5 to 100 mM may preferablybe used. More preferably, calcium ions at a concentration of 10 to 30 mMmay be used. In combination with a buffer to be used for adsorption toand elution from the cation exchange carrier, 20 mM ammonium chloridebuffer (pH 9.0) containing 20 mM calcium chloride may preferably beused.

The adsorbed calcium ion-binding protein may be eluted from the carrierby removing or decreasing calcium ion level in the buffer or addingother counter ions than calcium ions, or both. A counter ion includesNa⁺, Li⁺, K⁺ ions, and the like. Preferably, elution may be performed byadding 1 to 500 mM, more preferably 50 to 500 mM, still more preferably50 to 300 mM sodium chloride to the ammonium chloride buffer, and mostpreferably by adding 200 mM sodium chloride to 20 mM ammonium chloridebuffer (pH 9.0) containing 20 mM calcium chloride. Alternatively, theadsorbed calcium ion-binding protein may be eluted from the carriermerely by decreasing the calcium chloride level to less than 5 mM.

The method of the present invention, even when used solely, can affordto provide purification of a calcium ion-binding protein of 80% purityor more. It may more efficiently be used, however, in combination withother purification processes. For example, in case that a liquid samplecontaining a calcium ion-binding protein is contaminated with insolublesubstances, pretreatment for removing such substances, e.g.centrifugation, salting-out, membrane filtration, etc., is preferablycarried out prior to the method of the present invention.

In addition to the above-described processes, other purificationprocesses of various chromatographic procedures, including anionexchange chromatography, hydrophobic chromatography, gel filtrationchromatography, affinity chromatography, adsorption chromatography, etc.may be performed together with the method of the present invention toprovide a calcium ion-binding protein of higher purity. The method ofthe present invention may be used at any stage of the above-describedprocesses. Preferably, after a sample containing a calcium ion-bindingprotein is pretreated to remove insoluble substances, the method of thepresent invention is used and then anion exchange chromatography isfollowed. More specifically, an Annexin V-containing fraction obtainedby the cation exchange chromatography is applied to Q-Sepharose columnequilibrated with 10 mM sodium phosphate buffer (pH 7.4) containing 50mM sodium chloride, and after washing, elution is performed with lineargradient of concentration from 50 mM to 500 mM sodium chloride to giveAnnexin V with much higher purification.

A calcium ion-binding protein to be purified by the method of thepresent invention typically includes Annexins I, II, III, IV, V, VI andVII but may be any protein that has an ability to bind to calcium ions,such as coagulation factor X.

The method of the present invention may efficiently be applied to blood,body fluid and tissue homogenate from an animal either naturallyoccurring or genetically engineered that produces a calcium ion-bindingprotein, as well as cell homogenate and culture supernatant ofrecombinant cells, including plant cells, bacterial cells, yeast cells,insect cells and animal cells. preferably, the method of the presentinvention may be used in recombinant yeast cells producing a calciumion-binding protein. More preferably, the method of the presentinvention may be used in cell homogenate or culture supernatant of yeastcells producing Annexin V.

Annexin V thus prepared, having special physiological activities, may beformulated into a pharmaceutical preparation in any conventional dosageform such as injections, eye drops, oral preparations, suppositories,etc. alone or in combination with a pharmaceutically acceptable carrier,diluent, stabling agent or preservative.

According to the present invention, an efficient method for purifying acalcium ion-binding protein with high purity is provided. Also providedis the calcium ion-binding protein thus obtained by the method of thepresent invention having substantially no contaminants.

According to the method of the present invention, most proteinsnegatively charged under physiological conditions are passed through thecation exchange carrier whereas a calcium ion-binding protein, which canform a complex with calcium ions, is preferentially adsorbed to thecarrier. Thus, the method of the present invention enables handling of alarge quantity of a sample at one time without deterioration of theadsorption capacity of the cation exchange carrier by contaminatingproteins other than the desired protein.

EXAMPLE Preparation Example Preparation of Recombinant Yeast CellProducing Annexin V

Recombinant yeast cells producing Annexin V were prepared as describedin a publication of patent application (Japanese Patent Publication No.219875/1991). FIG. 1 schematically shows preparation of the recombinantyeast cells wherein the term “CPB-I” is used for referring to “AnnexinV”.

(1) Cloning of Annexin V Structural Gene

From human placenta cDNA library (Clontech Laboratories, Inc.), phagethat bears Annexin V structural gene was insolated by immunoscreeningusing anti-Annexin V monoclonal antibody. DNA was then prepared fromphage and digested with restriction enzyme EcoRI to produce a fragment,which was then inserted into the EcoRI site of pUC118 vector toconstruct pMKT7.

(2) Construction of Expression Plasmid

The plasmid pMKT7 was digested with restriction enzymes NcoI and SacIand a DNA fragment containing Annexin V structural gene was separated byagarose electrophoresis. Addition of a synthetic linker converted bothends of the DNA fragment into XhoI and BamHI sites. The resulting DNAfragment was inserted into XhoI and BamHI sites of the expression vectorpPS1 to construct expression vector pAPCPBI.

(3) Preparation of Recombinant Yeast Cells

Host yeast cells (Saccharomyces cerevisiae AH22) were transformed withthe expression plasmid pAPCPBI by the lithium acetate technique. Aftertransformation, colonies appeared on an agar medium deprived of leucinewere isolated and an expression level was measured. Those clones withhigher expression level were selected and subjected to repetition ofplating to the agar medium, isolation of colonies and measurement ofexpression level to give recombinant yeast cells with stability.

Example 1 Purification of Recombinant Annexin V

(1) Culture of Annexin V-Producing Recombinant Yeast Cells

Annexin V-producing recombinant yeast cells were cultured on 2Lsynthetic selection medium at 28° C. for 3 days. The recombinant yeastcells were then inoculated to 88L selection medium and cultured at 28°C. for 2 days. The recombinant yeast cells were then transferred to 810Lsemisynthetic medium (40 g sucrose, 5 g yeast extract, 5 g ammoniumsulfate and 0.5 g magnesium sulfate septahydrate in 1L medium) andculture was continued at 28° C. for 24 hours.

(2) Pretreatment of Annexin V in Large Quantity Prior to Purification

The large culture solution was filtered with a 0.1 μm membrane filter tocollect the recombinant yeast cells, which were physically ruptured witha French press-type cell homogenater. The ruptured cell suspension wasfiltered with the membrane filter and the filtrate was concentrated witha ultrafiltrater. To the concentrate was added acetic acid forisoelectric precipitation (pH 5.0). Precipitates formed were filteredwith the membrane filter to remove the precipitates. Then, pH of thefiltrate was adjusted to 9.0 with ammonia and the filtrate was againconcentrated with a ultrafiltrater (pretreated solution).

(3) Cation Exchange Chromatography (Elution by Decreasing or RemovingCalcium Chloride Level)

To the pretreated solution was added a calcium chloride solution to afinal concentration of 20 mM and was subjected to cation exchangechromatography with SP-Sepharose (Pharmacia). Specifically, thepretreated solution supplemented with calcium chloride was applied to acolumn equilibrated with 20 mM ammonium chloride buffer (pH 9.0)containing 20 mM calcium chloride and 50 mM sodium chloride. Afterwashing with the buffer, the column was further washed with 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride.Then, Annexin V was eluted with 20 mM ammonium chloride buffer (pH 9.0).

(4) Cation Exchange Chromatography (Elution by Increasing SodiumChloride Level)

As in the step (3), the pretreated solution was added with a calciumchloride solution to a final concentration of 20 mM and was subjected tocation exchange chromatography with SP-Sepharose. Specifically, thepretreated solution supplemented with calcium chloride was applied to acolumn equilibrated with 20 mM ammonium chloride buffer (pH 9.0)containing 20 mM calcium chloride and 50 mM sodium chloride. Afterwashing with the buffer, Annexin V was eluted by a linear gradient ofconcentration of sodium chloride from 50 mM up to 300 mM with 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride(flow rate at adsorption and elution: 56.7 cm/h).

FIG. 2 shows elution patterns obtained by gel filtration chromatographyof (a) sample prior to cation exchange chromatography, (b) fractionspassed through cation exchange chromatography, and (c) fractions elutedfrom cation exchange chromatography, respectively. Gel filtrationchromatography was performed wherein 20 μL sample was applied to TSKgelG3000 SW×1 (7.8 mm (ID)×30 cm) equilibrated with 10 mM phosphate buffer(pH 7.2) containing 0.14 M NaCl at a flow rate of 125.6 cm/h. Theresults of gel filtration chromatography for fractions eluted fromcation exchange chromatography are shown in Table 1 wherein purity wasobtained from the elution pattern.

(5) Anion Exchange Chromatography (Comparison With ConventionalTechnique)

Anion exchange chromatography was performed for the pretreated solution.The pretreated solution was applied to Q-Sepharose (Pharmacia) columnequilibrated with 10 mM sodium phosphate buffer (pH 7.4) containing 50mM sodium chloride. After washing, Annexin V was eluted by a lineargradient of concentration of sodium chloride from 50 mM up to 300 mM.The results of gel filtration chromatography for the eluted fractionsare shown in Table 1.

Annexin V obtained by the conventional technique and that obtained bythe method of the present invention were measured for theiranti-coagulating activity after further purification with additionalpurification processes. Measurement of anti-coagulating activity wasmade in the same manner as the quantification of sodium heparindescribed in the Japanese Pharmacopoeia (the 13th revision, p.900-901).Anti-coagulating activity was calculated wherein prolongation in timefor coagulation induced by 1 mg of standard sample (Annexin V purifiedby the conventional technique) was defined as one unit (U). As a result,no inactivation of Annexin V obtained by the method of the presentinvention was observed (Table 1). TABLE 1 Anion exchange Cation exchangechromatography chromatography Purity (%) 81.86 100 Activity (U/mg) 0.9to 1.0 0.9 to 1.0

Example 2 Purification of Recombinant Annexin V

A recombinant Annexin V was purified as in Example 1 except that 20 mMcitrate buffer (pH 6.0) containing 20 mM calcium chloride and 50 mMsodium chloride was used in place of 20 mM ammonium chloride buffer (pH9.0) containing 20 mM calcium chloride and 50 mM sodium chloride, and 20mM citrate buffer (pH 6.0) was used in place of a linear gradientconcentration of sodium chloride from 50 mM up to 300 mM with 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride, inthe cation exchange chromatography of the step (4). Flow rate was alsoaltered to 15.6 to 54.6 cm/h at adsorption and 39 cm/h at elution.

FIG. 3 shows an elution pattern obtained by gel filtrationchromatography of fractions eluted from cation exchange chromatography.

Example 3 Purification of Annexin VT From Placenta

One placenta (about 500 g) excepting the anion and an umbilical cord wassliced into pieces, washed with 2 L physiological saline, and mincedwith a meat grinder. After adding 400 mL of 50 mM Tris-HCl buffer (pH7.4) containing 5 mM calcium chloride, 0.1% Triton X-100 and 5 mMbenzamidine, the mince was homogenated with a whirling blender. Thehomogenate was centrifuged at 10,000 rpm for 20 minutes to collectprecipitates, which were again suspended in 300 ml of 50 mM Tris-HClbuffer (pH 7.4) containing 50 mM EDTA and homogenated. The homogenatewas again centrifuged at 10,000 rpm for 20 minutes and an extract ofsupernatant was recovered (about 300 mL), to which 63 g ammonium sulfatewas added to prepare a 30%-saturated solution of ammonium sulfate. Aftercentrifugation to remove precipitates, to the supernatant was added 54 gammonium sulfate (60%-saturated ammonium sulfate) and precipitatedAnnexin VI fraction was recovered. Annexin V could preferentially berecovered in precipitates formed when the salt concentration was raisedto 80% saturation of ammonium sulfate.

The precipitates formed with a 60%-saturated solution of ammoniumsulfate were dissolved in 50 mM Tris-HCl buffer (pH 7.4) and dialyzedagainst the same buffer. A dialyzed solution (80 mL) was adsorbed toDEAE-Toyopearl (3×20 cm) equilibrated with the same buffer. Afterwashing with the same buffer, elution was performed by a linear gradientfrom the same buffer (180 mL) to 50 mM Tris-HCl buffer (pH 7.4)containing 0.3 M NaCl (180 mL)(each fraction: 4 mL/tube). Annexin VI ofinterest was eluted in fractions No. 46 to No. 50. These Annexin VIfractions were dialyzed against 20 mM ammonium chloride (pH 9.0), towhich was added calcium chloride to make finally 20 mM ammonium chloridebuffer (pH 9.0) containing 20 mM calcium chloride. The dialyzed solutionwas adsorbed to SP-Sepharose FF (1.5×8 cm) equilibrated with 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride(FIG. 4; fractions No. 1 to No. 15). After washing with the same buffer,elution was performed with the same buffer supplemented with 0.5 M NaCl(FIG. 4; fractions No. 45 to No. 70). Samples at each stage wereanalyzed by non-reductive SDS-PAGE and the results are shown in FIG. 5.

Example 4 Purification of Coagulation Factor X

Commercially available coagulation factor X (about 1 mg) was dialyzedagainst 20 mM Tris-HCl buffer (pH 8.0), to which was added one tenthamount of 200 mM calcium chloride to make finally 20 mM Tris-HCl buffer(pH 8.0) containing 20 mM calcium chloride. The dialyzed solution wasadsorbed to SP-Sepharose FF (0.8×7 cm) equilibrated with 20 mM Tris-HClbuffer (pH 8.0) containing 20 mM calcium chloride. After washing with 20mM Tris-HCl buffer (pH 9.0) containing 20 MM calcium chloride, elutionwas performed with 20 mM Tris-HCl buffer (pH 9.0) containing 0.3 Mcalcium chloride. In all stages, 5 mM benzamidine was added (Sincebenzamidine per se exhibits UV absorption at A280, an elution pattern isnot explicit; FIG. 6). Fractions at each stage were analyzed bynon-reductive SDS-PAGE and the results are shown in FIG. 7.

1. A calcium ion-binding protein selected from the group consisting ofAnnexins I, II, III, IV, V, VI and VII having a purity of 80 to 100% asdetermined by gel filtration chromatographic analysis, obtained by amethod, comprising: contacting a sample containing said protein with acation exchange carrier in the presence of calcium ions to let theprotein be adsorbed to the exchange carrier; and eluting the adsorbedcalcium ion-binding protein from the exchange carrier by (a) decreasingor removing the concentration of said calcium ions (b) adding counterions other than said calcium ions or (c) both (a) and (b).
 2. A calciumion-binding protein selected from the group consisting of Annexins I,II, III, IV, V, VI and VII having a purity of 80 to 100% as determinedby gel filtration chromatographic analysis or SDS-PAGE analysis,obtained by a method, comprising: contacting a sample containing saidprotein with a cation exchange carrier in the presence of calcium ionsto let the protein be adsorbed to the exchange carrier; and eluting theadsorbed calcium ion-binding protein from the exchange carrier by (a)decreasing or removing the concentration of said calcium ions (b) addingcounter ions other than said calcium ions or (c) both (a) and (b).
 3. Acalcium ion-binding protein selected from the group consisting ofAnnexins I, II, III, IV, V, VI and VII having a purity of 80 to 100% asdetermined on a molecular size basis, obtained by a method, comprising:contacting a sample containing said protein with a cation exchangecarrier in the presence of calcium ions to let the protein be adsorbedto the exchange carrier; and eluting the adsorbed calcium ion-bindingprotein from the exchange carrier by (a) decreasing or removing theconcentration of said calcium ions (b) adding counter ions other thansaid calcium ions or (c) both (a) and (b).